Proteases or enzymes with proteolytic activity catalyze the cleavage of peptide bonds. Proteolytic reactions are fundamental to many key biological processes such as cell growth, cell death, blood clotting, matrix remodeling and immune defense. A large number of pathogens, including viruses, bacteria and multicellular parasites also use proteases to infect host cells, complete their life cycle and degrade the host immune system. Therefore, a large number of specific proteases are becoming attractive drug targets. For example, protease inhibitors are being developed for the effective management of AIDS (e.g. HIV-1 protease inhibitors such as Ritonavir, Nelfinavir or Saquinavir), HCV hepatitis (hepatitis C virus protease inhibitors), malaria, Chagas' disease and schistosomiasis (papain, plasmepsin and falcipain inhibitors). Protease inhibitors are also being tested for the treatment of hypertension (angiotensin-converting enzyme inhibitors), liver cirrhosis (caspase-8 inhibitors), Alzheimer's disease (beta site APP-cleaving enzyme inhibitors), autoimmune diseases (cathepsin S inhibitors), rheumatoid- or osteoarthritis and cancer (matrix metalloproteinase—MMP- and caspase-1 inhibitors) (see, e.g. Vandeputte-Rutten et al., (2002) Curr Opin Struct Biol. 12:704-708; and Docherty et al., (2003) Biochem Soc Symp. 70:147-161).
In the past two decades, almost 250,000 scientific articles reported research on proteases, indicating that research in this field is very dynamic. Recent success with HIV-1 protease inhibitors and other anti-protease drugs used to treat cancer or autoimmune diseases proved that proteolytic activities are attractive drug targets for modern medical therapy. Advances in genomic research provide an increasing number of potential ways to modulate protease activities and progress in bioinformatics now enable drug-discovery researchers to read and analyze large amounts of data in record time. The bottleneck for high-throughput drug screening is now at the level of bioassays. Many compounds initially identified using in vitro assays fail in later phases of drug development because they cannot be used in a biologically relevant environment.
An illustrative cellular process of interest that involves specific proteolytic reactions is apoptosis or programmed cell death. Programmed cell death (PCD) is a fundamental process for normal embryonic development and maintenance of adult tissue homeostasis (see, e.g. Metzstein et al. Trends Genet. 14, 410-416 (1998); Putcha et al. Cell Death Differ. 11, 38-48 (2004); and Danial et al. Cell 116, 205-219 (2004)). Studies in invertebrates such as C. elegans have provided important clues about the molecular mechanisms of PCD1. However vertebrate models allowing genetic screens and in vivo studies would facilitate research in this field because fundamental differences exist between invertebrate and vertebrate PCD pathways (see, e.g. Putcha et al. Cell Death Differ. 11, 38-48 (2004); and Danial et al. Cell 116, 205-219 (2004)).
Zebrafish is one promising vertebrate model to study PCD because it presents several features which made C. elegans a successful model in this field of research, including a rapid development, a transparent embryo and amenability to large-scale genetic screens (see, e.g. Haffter, P., et al. Development. 123, 1-36; and Driever, W. et al. Development 123, 37-46 (1996)). Higher gene conservation between teleosts and human is an advantage of zebrafish over C. elegans but the nematode is a relatively less complex organism exhibiting an invariant pattern of PCD which can be easily observed in vivo (see, e.g. Metzstein et al. Trends Genet. 14, 410-416 (1998)). In zebrafish, only terminal deoxynucleotide transferase-mediated dUTP nick-end labelling (TUNEL) on fixed preparations has revealed normal patterns of apoptosis so far (see, e.g. Cole et al. Dev. Biol 240, 123-142 (2001)).
There is a need in the art to understand and characterize the physiology of proteolytic processes such as those involved in programmed cell death. This need has increased markedly in recent years as artisans seek efficient bioassays that can, for example, determine the role of proteases in both normal cellular processes as well as in the context of specific diseases. Also needed are improved methods for targeted delivery of therapeutic agents to cells. The invention disclosed herein satisfies these and other needs.